MKG vol 39 no 1 Jan 2006 isi.pmd
32
The effect of caffeine on osteoblast proliferation after tooth
extraction in Wistar rats
Budi Yuwono
Department of Oral Surgery
Faculty of Dentistry Jember University
Jember - Indonesia
ABSTRACT
Caffeine is the most well-known substance which consumed by most people daily. Behind its popularity as favorable drinks and
food, this substance also known can inhibit the post extraction wound healing by decreasing the proliferation of osteoblast cells
through the increase of intracellular cyclic Adenosine Mono Phosphate (cAMP). The objective of this study was done to observe the
effect of caffeine intake toward the number of osteoblast cells during the wound healing of post dental extraction in Wistar’s rats.
This study was an experimental laboratory research and the post test-only control group design was used for the statistical evaluation.
The samples used were 24 healthy 3 months old male Wistar’s rats, with approximately 200 grams of body weight and devided into
4 groups. Three groups were taken and represented as a treated group (P) and the rest of one group was used as a control group
(KO). Caffeine diet with a dosage of 3.78 mg/100 ml grams of body weight/cc was given for 7 days in group P1, P2 for 14 days, and
21 days in group P3 and the diet was given orally using an oral sonde. Teeth extractions of the right first molar in the lower jaw were
done in all groups according to the interval time had been scheduled. Seven days of post-extraction time was waiting in all groups
before the sample being decapitated for further histological examination in the post extracted sites. A Hematoxillin and Eosin
staining was used and the number of osteoblast cells were counted under light microscopy with 400 times magnification. One-way
ANOVA and Least Significant Difference (LSD) test were used for the statistical evaluation. The result of the study shown a significant
decrease of the number of osteoblast cells in caffeine consumed group of 7, 14, and 21 days observed (p < 0.05). This study conclude
that the duration time of caffeine consumed had been interfered significantly with the osteoblast cell proliferation during the wound
healing after teeth extractions in Wistar’s rats.
Key words: caffeine, osteoblast, healing
Correspondence: Budi Yuwono, c/o: Bagian Ilmu Bedah Mulut, Fakultas Kedokteran Gigi Universitas Jember. Jln. Kalimantan I
No. 58 Jember 68121, Indonesia.
INTRODUCTION
Caffeine is the most well-known substance which is
consumed by approximately 80% people in the world. We
find this substance is in coffee, tea, chocolate, coke, ice
cream, yogurt, and orange juice. Caffeine is widely known
as a nerve stimulant and it is assumed to enhance
intelligence.1 Although caffeine is popular as favorable
drinks and food that consumed by children and adults daily,
caffeine can inhibit wound healing process. Based on our
experience, we found many patients with decreasing of
wound healing after dental extraction. This phenomenon
urged us to study further the role of caffeine in wound
healing process.
Kamagata et al.2 found that caffeine inhibit osteoblast
proliferation (in vitro) through the increasing of cyclic
Adenine Mono Phosphate (cAMP) as the cause of
intracellular phosphodiesterase inhibition. Furthermore, it
was reported that caffeine have a role in bone metabolism.
Wound healing process in lacerated tissue is initially
responded by an inflammation. The first change is in the
vascular stage by vasodilatating, extravasating and
exudating the wounded area. The release of Prostaglandin
E2 (PGE2) as the inflammation mediator in wound area
stimulate production of colagenase which trigger
degradation of collagen and other potent factor for bone
resorption, and also increase osteoclastic activity.3
Kamagata et al.2 reported that the increased PGE2 alone
could inhibit osteoblast proliferation through intracellular
cAMP increment (in vitro).
The released of PGE2 in wound area that accompanied
by certain doses of caffeine intake, could inhibit wound
healing. Kamagata et al.2 had proved that the combination
effect of caffeine and PGE2 could inhibit osteoblast
proliferation significantly (in vitro). More over, it was
reported that caffeine intake could increase PGE2
production and 0.1mM/ml caffeine could enhance PGE2
production.
Based on the explanation that caffeine intake at certain
doses and PGE2 can inhibit osteoblast proliferation and
increase osteoclast activity in bone resorption and
remodeling mechanism, it is assumed that caffeine is
influential towards the osteoblast number of post tooth
extraction.
33Yuwono: The effect of caffeine
The aim of this study is to evaluate the effect of caffeine
on the number of osteoblasts after tooth extraction wound
healing. Hopefully this study may develop the post tooth
extraction wound healing theory, give information about
substances which can inhibit the wound healing process
and also give information to patients who have high risk to
osteoporosis, diabetes mellitus, old age, stress and PMN
dysfunction caused by excessive caffein intake.
MATERIALS AND METHODS
This study was an experimental laboratory research with
a random PosT Test-only-control-group-design. The
samples were 24 healthy 3-month-old male Wistar’s rat,
with ± 200 grams of body weight and divided into 4 groups
(6 rats each). One group was control group with no caffeine
treated. Three groups (P1, P2, and P3) were treated with
3.78 mg/100gr of body weight/cc per oral caffeine (MERC,
Germany). Each group was placed in a different cage and
some food ad libitum were given. In control group: the
right molar in the lower jaw was extracted and was let live
for 7 days. Caffeine was given for 7 days in group P1, P2
for 14 days and 21 days in group 3, then the right molar in
the lower jaw was extracted and was let live for 7 days.
To obtain the right lower jaw, all samples were mortified
with ether anesthetic. Each right lower jaw was soaked to
10% formaldeyde to prepare an object/preparat of right
lower jaw bone with the routine procedure, decalcification,
dehydration and embedding. A sagittal section was prepared
showing post extraction dental socket. The sample was
7 section micrometer, painted and colored with a
Hematoxillin & Eosin routine dye, then with a light
microscope of 400x enlargement, the osteoblast cells were
counted in the post extraction socket. The data was analyzed
with One-way ANOVA and Least Significant Difference
(LSD) tests. The research was conducted at the biochemical
laboratory, Tropical Disease Center-Airlangga University
and at the pathologic laboratory of Dr.Soetomo General
Hospital in May until July 2005.
RESULTS
The average number of osteoblasts in post-extraction
wound healing could be seen in figure 1. There was an
average difference of the number of osteoblast in P1
(7 days), P2 (14 days) and P3 (21 days) as well as in the
control group.
To determined the differences of the osteoblast numbers
in 4 groups, a statistical test One-way ANOVA was
performed. The result have shown a significant difference
in group of control, P1 , P2, and P3 (p < 0.05). To find out
the difference of osteoblas numbers among groups, LSD
test with margin of error (α = 0.05) was performed. The
result showed a significant decrease of the number of
osteoblasts in all of the group (p < 0.05) as seen on table 1.
Table 1. LSD Analysis of inter group sample with degree of
error 0.05
Control
7 days
caffeine
14 days
caffeine
21 days
caffeine
Control
7 days caffeine
14 days caffeine
21 days caffeine
p = 0.001* p = 0.001*
p = 0.038*
p = 0.001*
p = 0.001*
p = 0.011*
Explanation: * = significant with α = 0.05
DISCUSSION
Based on many theories presented previously, it
revealed that caffeine intake could decrease osteoblast cells
activity due to the increased of PGE2 which was stimulated
by the enhancement of intracellular cAMP.
Caffeine will be absorbed by intestines, distributed to
all body parts including the nerve-circuit. The nerve
stimulant mechanism by caffeine was still unclear, but it
was found that the methyl compound of caffeine
substructure detached itself when interacted with cell
membrane. The methyl compound diffused between two
membrane layers, adding methyl component to its fat,
resulting a change in surface tension. The decreased of
surface tension made the membrane damped by water and
soluble substance easily, until the ions could easy to diffuse
and push ion formation effectively. It caused a more active
intersynapse exitation. The active exitation is the electrical
simulator for the release of central neurotransmitter like
Gamma Amino Butirate Acid (GABA) or choline acethyl
which is make strong post synaptic respond to stimulate
the central nervous system actively.4
The stimulation of the central nervous system trigger
the cell surface to release A2 phospholipase enzyme. The
A2 phospholipase pushed ahead the release of arachidonic
acid from the lipid membrane and converse it to PGE2 cyclic
derivative through the work of cyclo-oxygenase. The PGE2
give a certain effect to the body i.e. inhibiting electrolyte
resorption on proximal tubulus and make the entering water
was not re-absorbed, thus causing a constant excretion
quantity, yielding a tendency to diuresis.5
54.4
47.33
43.33
38.33
0
10
20
30
40
50
60
o
st
eo
b
la
st
co
u
nt
control caff. 7 days caff. 14 days caff. 21 days
Figure 1. Block diagram of the average osteoblast number in
the control group, 7 days caffeine, 14 days caffeine
and 21 days caffeine.
34 Maj. Ked. Gigi. (Dent. J.), Vol. 39. No. 1 January–March 2006: 32–34
Lerner and Mellstrom6 reported that caffeine has a role
on bone resorption in relation to calsium. Caffeine could
raise the spontaneous detachment of calsium mineral from
neonatal mouse calvarian bone (in vitro). A certain dose of
caffeine could trigger the raise of calsium detachment from
bone, which could stimulate bone resorption action through
cAMP cyclase adenilate system.
The increased of PGE2 not only bring a diuretic effect,
it also directly influence osteoclastic differentiation through
a raising activity of mononucleat precursor to become
mature osteoclast. Beside this activity, PGE2 also increase
mature osteoclast activity to re-absorb bone and to form
extracellular matrix.7 This effect was related to the increased
of cAMP inside the bone through activated cyclase
adenilate that increased the number, activity and
mobilization of osteoclast.8
High PGE2 level also decrease osteoblast differentiation,
by specific receptor which is raise phosphodiesterase
enzyme activity in osteoblast. Phosphodiesterase enzyme
stimulated cAMP degradation, lowering cAMP content,
resulting a decrease in osteoblast activity.7 Basically, PGE2
influenced bone changes to become more effective.
The increase of PGE2 level by caffeine that influenced
the escalation of osteoclasts activity will stimulate
bone resorption. This statement was in accord with
Kamagata et al.,2 that 0.1mM/ml caffeine could already
increase PGE2 production that inhibit osteoblast activity.
PGE2 also activated osteoclast which was crucial in bone
resorption.
Beside its popularity as favorable drinks and food,
caffeine was also assumed to inhibit wound healing process.
Caffeine inhibited osteoblast cell proliferation (in vitro),
through the increased level of cAMP caused by intracellular
phosphodiesterase.2 The increased cAMP level inhibited
osteoblast proliferation. Moreover, it was reported that
caffeine influence bone metabolism.
Wound healing process in lacerated tissue is initially
responded by an inflammation. The first change is in the
vascular stage by vasodilatating, extravasating and
exudating the wounded area. The release of Prostaglandin
E2 as the inflammation mediator in wound area, could
stimulate the production of collagenase enzyme production
which could trigger collagen dehydration, other potent
factor for bone resorption and could increase osteoclastic
activity.3 Kamagata et al.2 reported that the increased PGE2
alone inhibit osteoblast proliferation through intracellular
cAMP increment (in vitro).
The released PGE2 in wound area accompanied by
certain doses of caffeine intake could inhibit wound healing.
A study done by Kamagata et al.2 had proved that the
combination effect of caffeine and PGE2 could inhibit
osteoblast proliferation (in vitro). significantly Moreover,
it was reported that caffeine intake could increase PGE 2
and 0.1 mM/ml caffeine could already increase PGE2
production.
Grounded on the result of this study (in vitro), it has
been proven that caffeine can decline significantly the
number of osteoblast cell (p < 0.05) on 3 groups of 7, 14,
and 21 days caffeine diet. The longer the caffeine intake
take place, the more decreased of osteoblast cell numbers.
This fact strengthen the assumption that caffeine both in
vitro and in vivo is involved in bone mechanism, resorption,
apposition on wound healing process.
This research demonstrated that chronic caffeine intake
has a risk towards the decline of osteoblast cells. The
duration time of caffeine consumed had been interfered
significantly with the osteoblast cell proliferation during
the wound healing after teeth extractions in Wistar’s rats.
REFERENCES
1. Fen S. Kafein sebuah penenang semu. Jakarta: Penerbit Pustaka
Utama; 1993. p. 78–9.
2. Kamagata Y, Mitsuhiro C, Gina, Saltzman MJ. Combined effects of
PGE2 and caffeine on alveolar resorption. J Periodontology 1999
March; 15:234–38.
3. Nakano K, Ohishi M, Ogawa Y, Ohba T, Kido J. Protaglandin E2
inhibit alveolar bone resorption in experimental periodontitis in
Hamsters. J Periodontology 1998 March; 34:108–11.
4. Ungaro, Feas JKRN, Marry GR, Casey B. The effect of caffeine and
alcohol on hepatic cellular. J Medical & Health 1997 July; 34:65–9.
5. Guyton. Textbook of human psychology. London: Mosby-Year Book
Inc; 1995. p. 711–15.
6. Lerner UH, Mellstrom D. Caffeine has the capacity to stimulate
calcium release in organ culture of neonatal mouse calvaria.
University of Umea, Sweden. Calcif Tissue Int; 1992. p. 424-28.
7. Schwatz ZJ, Goultschin DD, Dean, Byan BD. Mechanism of alveolar
bone destructions of periodontitis. J Endodontic 1997 December;
22:677–80.
8. Offenbecher S, Heasman, Collins JG. Modulation of host PGE2
secretions as a determinant of periodontal disease expression.
J Periodontology 1993 August; 64:432–44.
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/NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken voor kwaliteitsafdrukken op desktopprinters en proofers. De gemaakte PDF-documenten kunnen worden geopend met Acrobat en Adobe Reader 5.0 en hoger.)
/NOR <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>
/PTB <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>
/SUO <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>
/SVE <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>
/ENU (Use these settings to create Adobe PDF documents for quality printing on desktop printers and proofers. Created PDF documents can be opened with Acrobat and Adobe Reader 5.0 and later.)
>>
/Namespace [
(Adobe)
(Common)
(1.0)
]
/OtherNamespaces [
<<
/AsReaderSpreads false
/CropImagesToFrames true
/ErrorControl /WarnAndContinue
/FlattenerIgnoreSpreadOverrides false
/IncludeGuidesGrids false
/IncludeNonPrinting false
/IncludeSlug false
/Namespace [
(Adobe)
(InDesign)
(4.0)
]
/OmitPlacedBitmaps false
/OmitPlacedEPS false
/OmitPlacedPDF false
/SimulateOverprint /Legacy
>>
<<
/AddBleedMarks false
/AddColorBars false
/AddCropMarks false
/AddPageInfo false
/AddRegMarks false
/ConvertColors /NoConversion
/DestinationProfileName ()
/DestinationProfileSelector /NA
/Downsample16BitImages true
/FlattenerPreset <<
/PresetSelector /MediumResolution
>>
/FormElements false
/GenerateStructure true
/IncludeBookmarks false
/IncludeHyperlinks false
/IncludeInteractive false
/IncludeLayers false
/IncludeProfiles true
/MultimediaHandling /UseObjectSettings
/Namespace [
(Adobe)
(CreativeSuite)
(2.0)
]
/PDFXOutputIntentProfileSelector /NA
/PreserveEditing true
/UntaggedCMYKHandling /LeaveUntagged
/UntaggedRGBHandling /LeaveUntagged
/UseDocumentBleed false
>>
]
>> setdistillerparams
<<
/HWResolution [2400 2400]
/PageSize [612.000 792.000]
>> setpagedevice